Micro-band electrode

ABSTRACT

The invention concerns an electrochemical cell which, either alone or together which a substrate onto which it is placed, is in the form of a receptacle. The electrochemical cell contains a working electrode and a counter electrode, the working electrode being in a wall of the receptacle. At least one of the electrodes has at least one dimension of less than 50 μm. The electrochemical cell is principally intended for use as a micro-electrode suitable for screening water, blood, urine or other biological or non-biological fluids.

FIELD OF THE INVENTION

The present invention relates to an electrochemical cell, typically amicro-electrode for electrochemical detection, a process formanufacturing such a cell and a method for electrochemically testing asubstance using the micro-electrode.

BACKGROUND TO THE INVENTION

Micro-electrodes are used for the electrochemical detection of variousparameters of a substance. For example, a micro-electrode may be used todetect, or measure the concentration of, a particular compound in a testsubstance. Typically, micro-electrodes contain an electrode which has atleast one dimension which is equal to or less than 50 μm, and frequentlya dimension of from 1 to 25 μm. The use of these systems as samplingdevices brings a number of potential benefits including speed ofoperation, accuracy and minimal sample requirement.

The common forms of large scale production fabricated micro-electrodesare either micro-disc, micro-band or interdigitated electrodes. Amicro-disc electrode is a plate like electrode with a diameter of lessthan about 25 μm whereas the micro-band electrode consists of a stripewith a thickness or smallest dimension of less than about 25 μm. Theinterdigitated electrode has a more complex form of two combs with theirteeth inter meshed.

By using these micro-electrodes in conjunction with enzymes or otherelectro-active substances it is possible to create sensors that providequantitative measurement of target parameters through reactions with thecorresponding electro-active substance.

However, several problems occur when using the micro-electrodes known inthe art in conjunction with an electro-active substance. Firstly,difficulties are frequently experienced in fixing the electro-activesubstance to the electrodes and movement of the substance away from itsdesired location is often seen. Systems containing severalmicro-electrodes on a single substrate are particularly susceptible toproblems in this regard, since enzymes which are not sufficientlyattached to their electrode become loose and migrate from one sensor toanother causing cross-contamination. This type of problem is exacerbatedby the effect of the sample flowing over the micro-electrode, whichtends to wash the electro-active substance off the electrode.

A common manner of immobilizing the electro-active substance, at leastto some extent, is to dry it in position on the electrode. However, thisis typically not sufficient to hold the electro-active substance inplace. Furthermore, drying the electro-active substance on top of themicro-electrode can cause electrical fouling of the electrode.

It is therefore an object of the present invention to provide amicro-electrode which is capable of holding an electro-active substanceat the electrode ready for sample testing and which will restrictmovement of any such electro-active substance whilst the sample flowsover the micro-electrode. It is also desired that the problems ofelectrode fouling which occur when an electro-active substance is driedto the electrodes will be avoided or reduced.

SUMMARY OF THE INVENTION

The present inventors have found that the problems discussed above canbe minimised when the micro-electrode is in the form of a receptacle.The receptacle comprises a working electrode in the wall of thereceptacle, typically having a small surface area. A counter electrodeis also present, this electrode typically having a much larger surfacearea than that of the working electrode, generally a surface area whichis at least an order of magnitude larger than that of the workingelectrode. The electro-active substance may be placed into thereceptacle and is optionally dried into position. The sample is thenapplied to the receptacle in order that testing can be carried out.

Such a micro-electrode is thus ideally suited to containing theelectro-active substance and preventing its movement away from theelectrodes. Furthermore, the effect of the sample flowing over themicro-electrode is much reduced and is unlikely to cause the enzyme tobe washed away from its position in the base of the receptacle.

The electro-active substance will typically not contact the workingelectrode in the wall of the receptacle during storage and thereforefouling of this electrode is minimised. Furthermore, the electro-activesubstance will typically contact only a small proportion of the counterelectrode and in some embodiments (discussed below) contact with thecounter electrode can be totally avoided. Therefore, if fouling doesoccur, this will only be to a relatively small area of the electrode.The remaining, unaffected areas of the counter electrode may stilloperate as normal.

Accordingly the present invention provides an electrochemical cellwhich, either alone or in combination with a substrate onto which it isplaced, is in the form of a receptacle, said cell comprising a counterelectrode and a working electrode, wherein at least one electrode has atleast one dimension not exceeding 50 μm, the working electrode being ina wall of the receptacle. The present invention in particular providesan electrochemical cell in the form of a receptacle, said cellcomprising a counter electrode and a working electrode, wherein at leastone electrode has at least one dimension not exceeding 50 μm, theworking electrode being in a wall of the receptacle.

The present invention also provides a process for producing anelectrochemical cell such as is described above, which process comprisesthe steps of:

-   -   (a) forming a first part comprising an insulating material which        is optionally coated with a counter electrode layer;    -   (b) forming a second part comprising a laminate of a working        electrode layer between two layers of an insulating material;    -   (c) creating a hole in the second part and    -   (d) bonding said first part to said second part to form a        receptacle.

Where a counter electrode layer is present in the first part, step (d)comprises bonding the counter electrode layer of said first part to saidsecond part to form a receptacle.

The process of the invention provides a simple and efficient way ofproducing the micro-electrodes of the invention. Furthermore, the stepof creating a hole in the part containing the working electrode mayeliminate the need for a separate step to activate the carbon, or otherworking electrode.

The present invention also provides a multi-analyte device whichcomprises a plurality of micro-electrodes in a single device. Thisdevice enables different types of measurement to be taken for a singlesample by using different electro-active substances in the variousmicro-electrodes. Alternatively, the multi-analyte device can be used tocarry out the same test on a single sample several times in order todetect or eliminate errors in results. The multi-analyte device of thepresent invention also ensures complete segregation of differentelectro-active substances since each micro-electrode is self-contained.

The present invention also provides a method of electrochemicallytesting a substance, the method comprising the steps of:

-   -   (a) insetting the sample into an electrochemical cell or        multi-analyte device of the invention;    -   (b) applying a voltage or a current between the working and        counter electrodes of the micro-electrode; and    -   (c) measuring the resulting current, voltage or charge across        the micro-electrode.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an electrochemical cell according to a first embodimentof the invention;

FIG. 2 depicts an electrochemical cell containing separate counter andreference electrodes in accordance with a second embodiment of theinvention;

FIG. 3 depicts an electrochemical cell having multiple workingelectrodes in accordance with a third embodiment of the invention;

FIG. 4 depicts an electrochemical cell having capiliary flow channels inaccordance with a fourth embodiment of the invention;

FIG. 5 depicts an electrochemical cell in which the counter electrode isin a wall or walls of the cell;

FIG. 6 depicts an alternative embodiment of the invention in which thecell itself is not in the form of a receptacle but forms a receptaclewhen placed on a substrate;

FIGS. 7, 8 and 9 show a multi-analyte device containing fourelectrochemical cells of the present invention;

FIG. 10 illustrates a process for producing the electrochemical cells ofthe invention;

FIG. 11 illustrates a modified process for producing the electrochemicalcells of the invention; and

FIGS. 12 to 20 illustrate the results of amperometric and cyclicvoltammetric experiments carried out using electrochemical cellsaccording to the invention.

DETAILED DESCRIPTION OF THE INVENTION

An electrochemical cell comprises a working electrode and a counterelectrode which are connected to one another electrically. When in use,electrochemical reactions occurring at each of the electrodes causeelectrons to flow to and from the electrodes, thus generating a current.An electrochemical cell can be set-up either to harness the electricalcurrent produced, for example in the form of a battery, or to detectelectrochemical reactions which are induced by an applied current orvoltage.

Embodiment 1

A first embodiment of the present invention is depicted in FIG. 1. Inthis embodiment, the electrochemical cell has a micro-electrode. Amicro-electrode has at least one dimension not exceeding 50 μm.Microelectrodes exhibit a typical microelectrode response when usingcyclic voltammetry. The microelectrodes of the invention may have one ormore dimensions which are macro in size, i.e. which are greater than 50μm. Due to these macro dimensions, the electrochemical cells of theinvention may exhibit some characteristics which are not usuallyassociated with microelectrodes. For example, the electrochemical cellsof the invention may exhibit some degree of Cottrell current. For thepurposes of the present specification therefore, the term microelectrodeis taken to include any electrode have at least one dimension notexceeding 50 μm.

Typically, the micro-electrode will be suitable for screening water(such as river water), blood, urine or other biological fluids orliquids such as beer and wine for determination of their contents. Thecell is in the form of a receptacle or a container. The receptacle maybe in any shape as long as it is capable of containing a liquid which isplaced into it. For example, the receptacle may be cylindrical.Generally, a receptacle will contain a base 1 and a wall or walls 2which surround the base. In one embodiment of the invention, which isdescribed further below, the cell itself does not have a base and thusis not, alone, a receptacle. However, the cell is designed such thatwhen placed against a separate substrate, the cell together with thesubstrate forms a receptacle. In this embodiment, the cell comprises awall or walls 2 which surround an open “base”. The open “base” may beplaced against the substrate to form a receptacle, such that thesubstrate forms the true base of the receptacle thus formed.

Typically, the receptacle will have a depth (i.e. from top to base) offrom 50 to 1000 μm, preferably from 200 to 800 μm, for example from 300to 600 μm. The length and width (i.e. from wall to wall), or in the caseof a cylindrical receptacle the diameter, of the receptacle is typicallyfrom 0.1 to 5 mm, for example 0.5 to 1.5 mm, such as 1 mm.

The open end of the receptacle 3 may be partially covered by animpermeable material as long as at least part of the open end isuncovered, or covered by a permeable material, such as a permeablemembrane. Preferably, the open end of the receptacle is substantiallycovered with a permeable membrane 4. The membrane 4 serves to preventdust or other contaminants from entering the receptacle, and helps tokeep any electro-active substance which might be inserted into thereceptacle in position.

The membrane 4 is preferably made of a material through which the sampleto be tested can pass. For example, if the sample is a blood sample, themembrane should be permeable to blood. Suitable materials for use as themembrane include polyester, cellulose nitrate, polycarbonate,polysulfone, microporous polyethersulfone films, PET, cotton and nylonwoven fabrics, coated glass fibres and polyacrylonitrile fabrics. Thesefabrics may optionally undergo a hydrophilic or hydrophobic treatmentprior to use. Other surface characteristics of the membrane may also bealtered if desired. For example, treatments to modify the membrane'scontact angle in water may be used in order to facilitate flow of thedesired sample through the membrane. The membrane may comprise one, twoor more layers of material, each of which may be the same or different.For example, conventional double layer membranes comprising two layersof different membrane materials may be used.

The membrane may also be used to filter out some components of thesample which are not desired to enter the cell. For example, some bloodproducts such as red blood cells or erythrocytes may be separated out inthis manner such that these particles do not enter the cell. Suitablefiltration membranes, including blood filtration membranes, are known inthe art. An example of a blood filtration membrane is Presence 200 ofPall filtration.

The electrochemical cell of the invention contains a working electrode 5which is situated in a wall of the receptacle. The working electrode is,for example, in the form of a continuous band around the wall(s) of thereceptacle. The thickness of the working electrode is typically from0.01 to 25 μm, preferably from 0.05 to 15 μm for example 0.1 to 20 μm,more preferably from 0.1 to 10 μm. Thicker working electrodes are alsoenvisaged, for example electrodes having a thickness of from 0.1 to 50μm, preferably from 5 to 20 μm. The thickness of the working electrodeis its dimension in a vertical direction when the receptacle is placedon its base. The working electrode is preferably formed from carbon,palladium, gold or platinum, for example in the form of a conductiveink. The conductive ink may be a modified ink containing additionalmaterials, for example platinum and/or graphite. Two or more layers maybe used to form the working electrode, the layers being formed of thesame or different materials. For example, a layer of Ag/AgCl may bepresent beneath the working electrode layer.

The counter electrode 6 typically forms at least a part of either thebase or the top of the receptacle, although the counter electrode mayalso be present in the wall or walls of the receptacle. In the presentembodiment, the counter electrode 6 forms the base of the receptacle.The counter electrode is typically made from Ag/AgSO₄ carbon, Ag/AgCl,palladium, gold, platinum, Cu/CuSO₄, Hg/HgCl₂ or Hg/HgSO₄. It ispreferably made from carbon, Ag/AgCl, palladium, gold, platinum,Cu/CuSO₄, Hg/HgCl₂ or Hg/HgSO₄. Each of these materials may be providedin the form of a conductive ink. The conductive ink may be a modifiedink containing additional materials, for example platinum and/orgraphite. Typically, the electrochemical cell of the invention containsonly one counter electrode.

The counter electrode 6 typically has a surface area which is of asimilar size to, or which is larger than, for example substantiallylarger than, that of the working electrode 5. Typically, the ratio ofthe surface area of the counter electrode to that of the workingelectrode is at least 1:1, such as at least 5:1, 10:1, preferably atleast 20:1, more preferably at least 25:1. The counter electrode may,for example, be a macroelectrode. Preferred counter electrodes have adimension of 0.01 mm or greater, for example 0.1 mm or greater. This maybe, for example, a diameter of 0.1 mm or greater. Typical areas of thecounter electrode are from 0.001 mm² to 10 mm², preferably about 5 mm².The minimum distance between the working electrode and the counterelectrode is preferably from 10 to 1000 μm, for example from 10 to 300μm.

In a typical cell according to the invention, each electrode will beseparated from the neighbouring electrode by a distance of from 10 to1000 μm, for example from 50 to 200 μm or from 75 to 150 μm. In orderthat the cell can operate, the electrodes must each be separated by aninsulating material 7. The insulating material is typically a polymer,for example, an acrylate, polyurethane, PET, polyolefin, polyester orany other stable insulating material. Polycarbonate and other plasticsand ceramics are also suitable insulating materials. The insulatinglayer may be formed by solvent evaporation from a polymer solution.Liquids which harden after application may also be used, for examplevarnishes. Alternatively, cross-linkable polymer solutions may be usedwhich are, for example, cross-linked by exposure to heat or UV or bymixing together the active parts of a two-component cross-linkablesystem. Dielectric inks may also be used to form insulating layers whereappropriate.

The electrodes of the electrochemical cell may be connected to oneanother and to any required measuring instruments by any suitable means.Typically, the electrodes will be connected to electrically conductingtracks which are themselves connected to one another and to the requiredmeasuring instruments.

The cell of the present invention may contain an electro-activesubstance 8. The electro-active substance 8 may be any substance whichis capable of causing an electrochemical reaction when it comes intocontact with a sample. Thus, on insertion of the sample into the celland contact of the sample with the electro-active substance,electrochemical reaction may occur and a measurable current, voltage orcharge may occur in the cell.

The electro-active substance 8 comprises an electrocatalyst. Typicallythe electro-active substance 8 comprises an electrocatalyst and amediator. A mediator is a chemical species that has two or moreoxidation states of distinct electro-active potentials that allow areversible mechanism of transferring electrons/charge to an electrode.The mediator reacts with the sample in the electrochemical reaction, thereaction being catalysed by the electro-catalyst. Typical examples of anelectro-catalyst are enzymes, for example lactate oxidase, cholesteroldehydrogenase, lactate dehydrogenase, glycerol kinase,glycerol-m-phosphate oxidase and cholesterol oxidase. Ionic species andmetal ions, for example cobalt, may also be used as the electrocatalyst.Examples of suitable mediators are ferricyanide, ferrocyanide andruthenium compounds such as ruthenium (III) hexamine.

The electro-active substance 8 is typically inserted into the receptaclein such a position that the electro-active substance is not in contactwith the working electrode. This ensures that fouling of the workingelectrode is minimised or avoided. The electro-active substance may bedried to ensure that it remains in position. In a preferred embodimentof the invention, the electro-active substance is pre-coated onto thesubstrate which forms the base of the receptacle. This may be doneeither by directly coating the electro-active substance onto a flatsubstrate, or by forming a well in the substrate and dispensing anelectro-active substance into the well. Typically, the electro-activesubstance is then dried into position and the thus-coated substrate isjoined to the walls of the receptacle. Where the electro-activesubstance is inserted into a well in the substrate, the well typicallyhas a cross-section which is identical to that of the finalelectrochemical cell. Thus, the well creates the bottom part of thereceptacle formed by the electrochemical cell. Where the counterelectrode is on the base of the receptacle, the electro-active substanceis pre-coated onto the counter electrode layer.

This embodiment has the advantage that the electro-active substance iskept remote from the working electrode at all times during manufactureof the cell. Contact between electro-active substance and workingelectrode is therefore minimised before the cell is used. This in turnminimises fouling of the working electrode.

In an alternative preferred embodiment, the electro-active substance isimpregnated into a membrane which is placed onto the substrate eitherbefore or after, preferably before, the substrate is joined to the wallsof the receptacle. The electro-active substance may equally beimpregnated into the membrane 4 which covers the cell. This embodimentalso avoids contact between the electro-active substance and the workingelectrode and minimises fouling.

The receptacle forming the micro-electrode of the present invention may,for example, contain one or more small air-holes in its base or its wallor walls (not depicted in FIG. 1). These holes allow air to escape fromthe receptacle when sample enters the receptacle. If such air-holes arenot present, the sample may not enter the receptacle when it flows overthe open end, or it may enter the receptacle only with difficulty. Theair holes typically have capiliary dimensions, for example, they mayhave an approximate diameter of 1-25 μm. Typically, from 1 to 4 airholes may be present.

Embodiment 2

A second embodiment of the invention, which is the same as the firstembodiment except as described below, is depicted in FIG. 2. In thisembodiment, the cell contains one or more reference electrodes 9 inaddition to the working and counter electrodes. In the case that noreference electrode is present (as in the first embodiment describedabove), the counter electrode acts as a reference or pseudo referenceelectrode. Typically, the reference electrode will be located in a wallof the receptacle 2. For example, the reference electrode may be in theform of a continuous band. The counter and working electrodes 6 and 5may be positioned such that the reference electrode 9 is located betweenthem, as is depicted in FIG. 2, or the counter and working electrodes 6and 5 may be adjacent. The reference electrode is typically made fromAg/AgSO₄, carbon, Ag/AgCl, palladium, gold, platinum, Cu/CuSO₄, Hg/HgCl₂or Hg/HgSO₄. It is preferably made from carbon, Ag/AgCl, palladium,gold, platinum, Cu/CuSO₄, Hg/HgCl₂ or Hg/HgSO₄. Each of these materialsmay be provided in the form of a conductive ink. The conductive ink maybe a modified ink containing additional materials, for example platinumand graphite.

Embodiment 3

A third embodiment of the invention, which is the same as either thefirst or second embodiments except as described below, is depicted inFIG. 3. This embodiment of the invention is a multi-ring electrode whichcontains one or more further electrodes 10, 10′ in addition to theworking, counter and optionally reference electrodes. The one or morefurther electrodes 10, 10′ typically act as additional workingelectrodes. Preferably, the counter electrode 6 acts as both the counterand the reference electrode and a separate reference electrode, asdescribed in embodiment 2, is not present.

Typically, the receptacle comprises no more than 10 electrodes in total,including working, counter and reference electrodes. Preferably no morethan 7 electrodes, more preferably no more than 5 electrodes arepresent. More preferred receptacles contain 2, 3 or 4 electrodes. Wheremore than one working and/or reference electrode is present, these aretypically located one above the other in the wall(s) of the receptacle.

The additional working electrodes, 10, 10′ allow different measurementsto be carried out simultaneously on the same sample by applyingdifferent potentials across two or more of the working/counter electrodepairs. Alternatively, the same potential may be applied to each workingelectrode and the same measurement recorded several times for the samesample. This helps to eliminate or detect errors in the measurementstaken.

In one particular example of this embodiment, one of the workingelectrodes is present on the base of the receptacle, i.e. in theposition in which the counter electrode 6 is depicted in FIG. 3. In thiscase, the counter electrode is present either in the wall(s) of thereceptacle as described below with reference to embodiment 5, or in thetop of the receptacle as described below with reference to embodiment 4.

Embodiment 4

A fourth embodiment of the invention, which is the same as the first,second or third embodiments except as described below, is depicted inFIG. 4. In this embodiment, the cell comprises one or more capiliarychannels 11 to allow sample to enter the receptacle. The capiliarychannels are, for example, covered by a capiliary film. Examples ofsuitable capillary films are PET films such as Melinex or ARcare®,adhesive coated films by Adhesive Research, and hydrophilic coated filmssuch as ARcare® 8877, which can offer better capillary performance. Inthis embodiment, the receptacle is preferably covered by a substantiallyimpermeable material 12. The impermeable material 12 is typically acapiliary film as described above. One or more capiliary channels 11 areprovided, for example in a wall or walls of the receptacle 2, throughwhich the sample may enter the receptacle. Typically, as is depicted inFIG. 4, the capiliary channel 11 is located at the point where the wall2 meets the impermeable material 12.

In order that air can escape from the receptacle and allow the sampleliquid to enter, one or more air holes must be present in thisembodiment. Typically, an air hole will be positioned at the point wherethe base meets the wall of the receptacle, as indicated by the label 12a in FIG. 4. The air hole(s) preferably have the dimensions describedabove and preferably from 1 to 4 air holes are present.

This embodiment has the advantage that the top of the receptacle isclosed and thus the counter electrode may either be located at the top3, at the base 1; or in the wall(s) 2 of the receptacle. The counterelectrode 6 is depicted at the top of the receptacle in FIG. 4. This isachieved by bonding the counter electrode 6 to the impermeable material12 prior to its attachment to the receptacle. In this way, theelectro-active substance 8, which is typically located on the base 1 ofthe receptacle, is not in contact with either the working or the counterelectrodes and thus electrode fouling is significantly reduced oreliminated. It is possible to locate the electro-active substance on thetop 3 of the receptacle, typically by pre-coating the substance onto thesubstrate which is to form the top 3 prior to its attachment to thereceptacle.

A further advantage of placing the counter electrode at the top of thereceptacle is that the base of the receptacle may be coated, or adaptedin another way, to make it more suitable to receive the electro-activesubstance which is typically dried onto the base. For example, the basemay be made of a particular material, such as carbon (provided that thecarbon is electrically insulated from the electrodes), which is suitablefor depositing enzymes on. Alternatively, the base may be coated with ahydrophilic coating.

If desired, the base of the cell may be formed from a permeable membranewhich may be of the same type as the membrane 4 discussed above. Themembrane is typically impregnated with an electro-active substance priorto, attachment to the cell. This avoids electrode-fouling caused bycontact between electro-active substance and working electrode duringinsertion of the electro-active substance.

Embodiment 5

An alternative embodiment of the invention is depicted in FIG. 5. Thisembodiment is the same as any one of embodiments 1 to 4 discussed aboveexcept as described below. The counter electrode 6 in the cell of thisembodiment is located in a wall or walls 2 of the receptacle. Thecounter electrode is, for example, in the form of a continuous bandaround the wall(s) of the receptacle.

The thickness of the counter electrode in this embodiment is typicallyfrom 0.1 μm to 1 mm, preferably from 5 to 500 μm, for example from 5 to100 μm, more preferably from 5 to 50 μm. The thickness of the counterelectrode in this embodiment is its dimension in a vertical directionwhen the receptacle is placed on its base. The ratio of the surface areaof the counter electrode to that of the working electrode may, in thisembodiment, be less than the preferred value of 25:1 which applies forcounter electrodes located in the base or top of the receptacle.Preferred ratios for this embodiment are in the range 1:1 to 10:1,preferably 2:1 to 5:1.

Embodiment 6

A further embodiment of the invention, which is depicted in FIG. 6,relates to a modification of the above described electrochemical cell inwhich the receptacle is completed when the cell is placed on a substrate21. In this manner; the substrate 21 forms the base of the receptacle.The cell of this embodiment alone is not necessarily in the form of areceptacle since it has an opening at the position of the base 1.However, when placed onto a separate substrate 21, the cell togetherwith the substrate forms a receptacle.

This embodiment therefore relates to a electrochemical cell comprising acounter electrode and a working electrode, wherein at least oneelectrode has a dimension of less than 50 μm, and wherein the cell has ashape such that, when placed on a substrate, the cell, together with thesubstrate on which it is placed, forms a receptacle, the workingelectrode being in a wall of the receptacle.

The electrochemical cell of this embodiment is at least partially openat its base 1. In this context, the term “open” includes a total absenceof a base material and also the presence of a material which allowssample liquid to pass through it. Typically, the cell's base 1 is eitherat least partially uncovered or at least partially covered with apermeable membrane. The permeable membrane is optionally impregnatedwith an electro-active substance prior to its attachment to the walls ofthe receptacle.

The top of the cell 3 may be totally or partially covered with apermeable membrane 4 (as depicted) or with an impermeable material. Ifthe cell is at least partially covered with an impermeable material, thecounter electrode may be located at the top of the cell coated onto theimpermeable material as described with reference to embodiment 4 above.This is achieved by bonding the counter electrode 6 to the impermeablematerial 12 prior to its attachment to the walls of the receptacle. Anelectro-active substance (not depicted), as described above, may also bebound to, the counter electrode prior to its attachment to the walls ofthe receptacle. If the cell is totally covered with an impermeablematerial, air-holes, as described with reference to embodiment 4, arepreferably present in order to facilitate entry of a sample liquid intothe cell. Alternatively, the counter electrode 6 may be located in thewall(s) of the cell as described in embodiment 5 above and as depictedin FIG. 6. Where the top of the cell 3 is open or covered only with apermeable membrane, the counter electrode is located in the wall(s) ofthe cell as here depicted.

The sample liquid to be tested enters the cell either through the top ofthe receptacle (where the top is not totally covered by an impermeablemembrane), or, more usually, through the open base. This is typicallyachieved by placing the cell onto a substrate which is already coatedwith the sample liquid. Alternatively, the cell may be placed onto thesubstrate, either directly or through a permeable membrane, and thesubstrate then pierced within the receptacle in order to introduce aliquid sample present under the substrate surface into the receptacle.For example, the substrate may be the skin and the sample liquid blood.Alternatively, the substrate may be a pre-packaged container in which asample liquid is present, the sample liquid being released when thecontainer is pierced.

A non-limiting example of a use of the cell of this embodiment is as aself-testing blood analysis kit. A diabetic user for example mightemploy such a cell to carry out glucose analysis tests on samples oftheir blood. This can be done by (i) piercing the skin, e.g. a finger,which is optionally covered with a permeable membrane, (ii) placing thecell over the blood spot produced such that the skin, or the permeablemembrane as relevant, together with the cell form a receptacle, and(iii) operating the cell in the usual manner. Alternatively, the cellmay first be placed onto the skin or permeable membrane and the skinsubsequently pierced through an open part of the receptacle. In the samemanner, the cell can also be used for other types of blood test.

The remaining features of the cell are typically as described above withregard to the other embodiments of the invention.

Multi-Analyte Device

The present invention also provides a multi-analyte device whichcomprises two or more micro-electrodes of this invention, for example inaccordance with any one of embodiments 1 to 6 above. Themicro-electrodes of the multi-analyte device may each be of the same ordifferent designs. Typical multi-analyte devices according to theinvention are described in FIGS. 7, 8 and 9. The multi-analyte devicewill typically comprise a plate or strip 14 which contains one or moremicro-electrodes 13 a, b, c and d. Each micro-electrode may contain thesame or different electro-active substances such that when a sample isinserted into each receptacle, several different tests may be carriedout or the same test may be repeated several times in order to detect oreliminate errors in the measurements taken. Furthermore, themicro-electrodes may be set at different potentials, again providingdifferent measurements for the same sample.

The micro-electrodes are typically separated by a distance of from 250μm to 550 μm, for example from 250 μm to 425 μm.

A multi-analyte device can also be made with a “vertical” arrangement ofcells as an alternative to Embodiment 3.

In this arrangement the sample in the first micro-electrode passes to afurther micro-electrode below it, for example, using a permeablemembrane in the base of the first micro-electrode, for a determinationof a different component in the sample. The permeable membrane may beimpregnated with an electro-active substance.

The electrical tracks 15 of the multi-analyte device are typically onthe top surface of the device. Filled vias are used to connect thecounter, optional reference and working electrodes to the surface tracks15 which then mate with a measuring instrument 16, or the laminatedback/counter can be arranged to mate with the instrument directly.

The multi-analyte device may contain one or more blank electrodes 17 asis depicted in FIG. 8. The blank electrode(s) do not contain a counterelectrode. This embodiment may, for example, be useful where theelectro-active substance has a working potential which conflicts withthat of the counter electrode system. In this situation, reduction oroxidation of the mediator contained in the electro-active substance mayoccur. Thus, for example, where the counter electrode is a Ag/AgClcouple and the mediator is ferricyanide, the redox state of the mediatoris such that it interacts with the Ag/ACl, forming a battery system orgalvanic cell in which reactions occur spontaneously as soon as there isliquid connection between them.

The multi-analyte device may also comprise capillary channels 18 as aredepicted in FIG. 9. These capillary channels are preferably of the typedescribed in embodiment 4 above. Thus, each receptacle is provided witha capillary channel which may optionally be connected to a singlechannel from which the sample is drawn.

Process for Producing Electrochemical Cells

A process for producing the electrochemical cells of the firstembodiment of the present invention is depicted in FIG. 10. The cellsmay be produced by a process which comprises the steps of:

-   -   (a) forming a first part 18 comprising an insulating material 18        a which is optionally coated with a counter electrode layer 18        b;    -   (b) forming a second part 19 comprising a laminate of a working        electrode layer 19 a between two layers 19 b and c of an        insulating material;    -   (c) creating a hole 19 d in the second part; and    -   (d) bonding said first part 18 to said second part 19 to form a        receptacle.

The materials, dimensions and other properties of the electrochemicalcell are as described above.

Where the counter electrode is in the base of the receptacle, the firstpart comprises an insulating material 18 a which is coated with acounter electrode layer 18 b as depicted in FIG. 10. In this case, step(d) comprises bonding the counter electrode layer 18 b of said firstpart 18 to said second part 19 to form a receptacle. Alternatively, whenthe counter electrode is in a wall or walls of the electrode asdescribed in embodiment 5 above, the counter electrode layer may beabsent from the first part and the second part comprises a counterelectrode layer between two layers of insulating material.

Step (c) in which a hole is created in the second part may be carriedout by any suitable means. For example, the hole may be punched ordrilled or formed by die-cutting, ultra-sonic cutting or laser drilling.This step has the advantage that the electrode surfaces areautomatically cleaned by the action of creating the hole, thus reducingthe requirement for a separate step of cleaning the electrodes.

A suitable technique for creating the hole is to punch the second partwith a pneumatic or hydraulic press tool. Holes of 0.1 to 5 mm,preferably 0.5 to 1.5, more preferably about 1 mm diameter arepreferred. The hole should extend down through all of the printed layersand the substrate. The punching tool can be coated with hardeningmaterials such as titanium and may or may not have an angled cuttingedge. For example, the tool may be Ti coated with a 2° angle from thehorizontal cutting edge.

The bonding step (d) may be carried out by any suitable bondingtechnique. For example, bonding may be performed using pressurizedrollers. A heat sensitive adhesive may be used, in which case anelevated temperature is needed. Room temperature can be used forpressure sensitive adhesive.

If desired, air channels may be created in the micro-electrode at thejoint between the first part 18 and the second part 19. This can beachieved, for example, by creating grooves in either the bottom side ofthe second part 19 b or the top side of the first part 18 a prior tobonding these two parts together.

Carbon or other inks may, for example, be printed onto the insulatingmaterial 18 a, 19 b, 19 c using a screen printing, ink jet printing,thermal transfer or lithographic or gravure printing technique, forexample the techniques described in GB 6106417.9. The insulating layer19 c may also be formed by printing an insulating material onto theworking electrode layer. Other techniques for forming the insulatinglayer include solvent evaporation of a solution of the insulatingmaterial or formation of an insulating polymer by a cross-linkingmechanism.

Each electrode is typically printed, or otherwise coated, onto therelevant insulating layer in a chosen pattern. For the working electrodeor other electrodes which are to be formed in the wall of thereceptacle, the pattern selected should be such that at least a part ofthe electrode layer is exposed when hole 19 d is created. Preferably thepattern chosen is such that the electrode layer is exposed around thewhole, perimeter of hole 19 d.

In one embodiment, two or more printing or other coating steps arecarried out to create an electrode layer. One or more steps, preferablyone step, uses a pattern which deposits conductive material in the areawhich will form the perimeter of hole 19 d as well as, for example,areas which are to form conductive tracks. This layer is exposed whenhole 19 d is created and forms the electrode. One or more further stepsuses a pattern which deposits conductive material, for example, in areaswhich are to form conductive tracks but deposits no material in the areawhich will form the perimeter of hole 19 d. These areas are not exposedwhen hole 19 d is made. Thus, a thin electrode layer is formed aroundhole 19 d, leading to a thin electrode in the wall of the finishedreceptacle, whilst a thicker layer is formed away from hole 19 d. Thisthicker layer has a lower resistance and thus leads to a more efficientfunctioning of the electrochemical cell. This use of a double layer isparticularly preferred with regard to the working electrode.

If desired, the one or more layers may be formed of different materials.For example, the layer which will be exposed at hole 19 d may be formedof carbon whilst a further layer, for example a sub-layer, of adifferent material may be used.

The working electrode, counter electrode and reference electrode may allbe produced by printing ink containing the desired material onto thesubstrate. Insulating layers may also be produced in this manner byprinting an ink containing an insulating material onto a substrate oronto a conductive layer. Screen printing is a preferred manner in whichthis is carried out. Typically, a conductive layer will be printed ontoa substrate and a dielectric layer will be printed onto the conductivelayer.

Screen printing is generally carried out on polyester, polycarbonate, orother plastic/ceramic substrate. Types of substrates used are forexample, DuPont films of Mylar A, Mylar ADS, Melinex, Kaladex, TejinTetoron, Purex, Teonex. Substrates used are preferably surface treatedto improve adhesion of the ink to the substrate, for example by coronadischarge or chemical modification. Substrates are also preferablylaminated on one side, for example with either heat sensitive orpressure sensitive adhesive in the thickness range 20 μm to 200 μm,preferably about 40 μm. A preferred embodiment employs 250 μm thickMylar ST535 with 40 μm thermally activated adhesive laminate as asubstrate.

A screen is selected from stock with the carbon stencil defined withphotosensitive emulsion with a thickness of 10 μm to 20 μm, preferablyabout 13 μm. The required thickness of the print is determined by themesh count of the screen. Typically this is within the range of 83t/inch to 330 t/inch, preferably 305 t/inch for both carbon Ag/AgClinks, and about 195 t/inch for dielectric ink. The ink is typicallyforced through the mesh using a squeegee rubber of 65 to 85 shorehardness, preferably 75-shore hardness.

Suitable mesh counts are as follows:

-   Approx thickness of print when using 330 t/inch/=7 μm    -   305 t/inch/120 t/cm=10 μm    -   195 t/inch/77 t/cm=15 μm    -   156 t/inch/61 t/cm=20 μm    -   83 t/inch/=25 μm

The printed layer is typically dried using the ink manufacturerrecommendations. It is typically stoved in an oven for 2 minutes to 4hours, preferably 1 hour, at about 70-130° C. Air drying, or air forcedtunnel drying for 2-3 minutes at 90-130° C. may also be used.

The screen printed dielectric layer can be replaced by a laminate ofpolyester, polycarbonate or similar (preferably Mylar ST535) whichcovers the carbon layer and with thickness in the range 10 μm to 200 μm,preferably 10 μm to 30 μm.

Suitable inks for use in the screen printing processes are as follows:

Carbon Inks:

-   1. Coates carbon 26-8203-   2. Ercon G449-   3. Du-pont L881    Dielectric Inks:-   1. Ronseal ultra tough hardglaze clear varnish.-   2. Ercon E6165-116 blue insulator.-   3. Du-Pont 5036 encapsulant-   4. Coates screen flex coverlay.    Silver/Silver Chloride Inks:-   1. Gem ag/agcl-   2. Ercon E0430-128-   3. Du-Pont 5874 conductor

After forming the receptacle, an electro-active substance as describedabove may be inserted into the micro-electrode, for example, usingmicropipetting or enzyme jet printing. The electro-active substance maythen be dried by any suitable technique. Alternatively, theelectro-active substance may be pre-coated onto the base before bondingstep (d) takes place. To achieve this, the electro-active substance istypically coated onto layer 18 b, dried into position and subsequentlyparts 18 and 19 are bonded together as described above. A further optionis to impregnate the electro-active substance into a membrane which canbe placed on, or fixed onto, layer 18 b prior to or after bonding step(d).

If desired, a permeable membrane may then be placed over the receptacle(as in FIG. 1). Membrane structures are applied to the top surface ofthe device using double sided adhesive or screen printed pressuresensitive adhesive. Attachment of the membrane 20 may, for example, becarried out by using a pressure sensitive adhesive (which has been cast)that has been die cut to remove the adhesive in the area over thereceptacle. In the embodiments in which the electro-active substance isimpregnated into membrane 4, impregnation of the desired substance istypically carried out before the membrane is attached to the receptacle.

If one or more capiliary channels are desired, these are preferablyformed by creating one or more grooves in the top of the second part 19c, the grooves being connected to the hole 19 d, or the top of thereceptacle. The grooves may conveniently be created during the sameprocess as creating the hole in the second part. For example, using atechnique of pressing, punching, die-cutting, ultra-sonic cutting orother suitable film fabrication technique. The second part may then becoated with an impermeable material, for example a capiliary film asdescribed above, thus creating a capiliary channel connected to thereceptacle and which allows a sample to enter the receptacle.

A modified process may be used when the electro-active substance is tobe pre-coated into a well in the substrate which forms the base of thereceptacle. This modified process is depicted in FIG. 11.

In this process, step (a) comprises, if desired, coating insulatinglayer 18 a with counter electrode layer 18 b as described above. Afurther insulating layer 18 c is provided which has a pre-formed hole 18d. Hole 18 d is typically of the same size as hole 19 d and may beformed by the techniques mentioned above with reference to hole 19 d.Insulating layer 18 c is bonded to layer 18 b thus creating a well inthe position of hole 18 d. An electro-active substance is then dispensedinto this well, for example using micro-pipetting or enzyme jetprinting. The electro-active substance may then be dried by any suitabletechnique. Following addition of the electro-active substance, Part B(18) may be used in bonding step (d) in the manner described above.

An alternative process may be used when the invention is to be producedin accordance with embodiment 4 above. In this embodiment, the processcomprises the steps of:

-   -   (a) forming a first part comprising an insulating material;    -   (b) forming a second part comprising a laminate of a working        electrode layer between two layers of an insulating material;    -   (c) creating, in the second part, a hole and a capiliary channel        to allow a sample to enter said hole;    -   (d) bonding said first part to said second part to form a        receptacle;    -   (e) placing an electro-active substance as described above into        the receptacle and optionally drying the electro-active        substance; and    -   (f) bonding to the open end of said receptacle a layer which is        optionally coated with a counter electrode material.

The materials, dimensions and other properties of the electrochemicalcell are as described above. Step (c), comprising forming a hole and acapiliary channel in the second part may be carried out as describedabove. In this process, the impermeable material or capiliary film istypically coated on the underside with a counter electrode materialbefore it is bonded. Thus, when this layer is coated to the top of thereceptacle, a counter electrode is formed. Alternatively, when thecounter electrode is in a wall or walls of the electrode as described inembodiment 5 above, the counter electrode layer may be absent from thelayer used in step (f) and instead the second part comprises a counterelectrode layer between two layers of insulting material.

In a modification of the above process, the electro-active substance maybe pre-coated onto either the base or the top of the receptacle by anydesired technique, for example those discussed above, therebyeliminating the need for step (e). Thus, an alternative preferredprocess comprises steps (a), (b), (c), (d) and (f) above and employs (i)a first part which comprises an electro-active substance on its surfaceand/or (ii) a layer for use in step (f) comprising an electro-activesubstance its surface. Where said layer for use in step (f) comprises acounter electrode layer, the electro-active substance is typicallycoated onto the counter electrode layer. Following bonding step (f), areceptacle is thus formed having an electro-active substance coated tothe interior surface of either the base or the top. If desired, theelectro-active substance may be placed into a well in the substrate asdiscussed above in relation to FIG. 11.

In one embodiment, the insulating material of the first part is apermeable membrane as described above. The membrane is optionallyimpregnated with an electro-active substance prior to bonding step (d).

In order to form the electrochemical cell described in Embodiment 6above, a modified version of any of the above described processes isused in which the step of bonding the first part to the second part isomitted. Thus, the process comprises:

-   -   (a) forming a second part 19 comprising a laminate of a working        electrode layer 19 a between two layers 19 b and c of an        insulating material,    -   (b) creating a hole 19 d in the second part; and optionally    -   (c) bonding to said second part a layer which is coated with a        counter electrode material.

Steps (a) and (b) are carried out as described above with reference tocorresponding steps. The process may optionally comprise a further step,which may be carried out before or after step (c), of attaching to thebottom of the second part a permeable membrane which may optionally havean electro-active substance impregnated into it. If the counterelectrode is present in the top of the cell, step (c) above is carriedout. If the counter electrode is present in a wall of the cell, step (c)may be omitted and the second part additionally comprises a layer ofcounter electrode material between two layers of insulating material.

An electro-active substance may be coated onto the counter electrodelayer, if desired and alternatively or additionally to theelectro-active substance bound to any permeable membrane present. Thiscoating process may be carried out as described above.

In order to form the multi-analyte devices of the present invention, thestep (c) described in one of the two processes above is extended toinclude the formation of two or more holes in the second part. Thus,when the bonding step (d) is carried out, two or more receptacles areformed. Where capiliary channels are used, these may be formed asdescribed above at each of the receptacles. Thus, samples may be drawninto each micro-electrode by capiliary action.

Typical Uses of the Electrochemical Cell

The electrochemical cell of the present invention is intendedprincipally for use as a micro-electrode for screening purposes, i.e.for screening liquid samples. For example, the cell may be used fordetermining the content of various substances in water, beer, wine;blood or urine samples, or samples of other biological or non-biologicalfluids. The cells may, for example, be used to determine thepentachlorophenol content of a sample for environmental assessment; tomeasure cholesterol, HDL, LDL and triglyceride levels for use inanalysing cardiac risk, or for measuring glucose levels, for example foruse by diabetics. A further example of a suitable use for the cells ofthe invention is as a renal monitor for measuring the condition of apatient suffering from kidney disease. In this case, the cells could beused to monitor the levels of creatinine urea, potassium and sodium inthe urine.

Whilst the major use envisaged for the electrochemical cells of theinvention is as a microsensor, the cells may also be used for any otherpurpose in which electrochemical measurement or the harnessing ofelectrochemical energy takes place. For example, the electrochemicalcell of the invention may be used as a battery. The cell may also beused to process an electro-active substance such as an intercalatingmaterial used for detection of electrolytes such as sodium, potassium,calcium and phosphates. Such processing may involve electro-cycling ofthe substance in order to develop a consistent thin layer on theelectrodes.

EXAMPLES Example 1 Manufacture of Electrochemical Cell

A base film of 125 μm thick PET was printed with the counter/referenceelectrode using a silver/silver chloride printing ink, and then dried at90° C. for 30 minutes.

A middle film of 250 μm PET was coated with heat seal. The film was thenprinted on the reverse side to the heat seal coating with a conductivecarbon ink in a pattern that defines the conductive tracks. This wasthen dried at 90° C. for 1 hour. The carbon ink print was subsequentlyoverprinted with a dielectric ink, except for the part of the tracksthat were required to mate with the connector in the measuringinstrument, where over printing was not carried out. The dielectric inkwas then dried at 60° C. for 20 minutes.

Several holes were then formed in the middle layer using a punch thatforms the holes using a shearing action. This punch comprised metal diesor pins having a diameter equal to that of the required holes. The metaldies or pins were used to shear the film which was supported by metal orwooden plates having holes that match the formation of the punch inorder to allow the punch to slide.

Following punching of the holes, the middle film was laminated to thebase film using heat. During the heating step, the heat seal on theunderside of the middle film melts and bonds to the base film.

The desired electro-active substances were then dispensed into the wellsformed. The substances were then dried using room temperature airflowover the surface.

Over some of the wells, a blood separation membrane was added that iscapable of removing the larger cellular particles from whole blood. Forthese electrodes, a blood separation membrane such as Presence 200 byPall filtration was attached to the top most surface of the electrodescovering the wells. Attachment of the membranes was accomplished byusing a screen printed pressure sensitive adhesive cast around the wellsonto the middle layer.

Example 2 Use of Electrochemical Cell

Electrodes were constructed from a 250 μm PET layer on which a 15 μmCoates carbon ink 26-8203 layer had been screen-printed followed by a 30μm layer of Ronseal ultra tough hardglaze clear varnish (a polyurethanebased on Baxenden trixine containing polyurethane and isocyanates). Thislayer was punched to produce a 1 mm diameter hole. A PET base layer wasproduced consisting of a 125 μm PET layer having a common Ag/AgClcounter reference on the top. The PET base layer was then adhered to thepunched layer using ARcare 7841 sheet adhesive. Various tests werecarried out using this electrochemical cell as described below atExamples 2a to 2f.

Example 2a

Cyclic voltammetric current was measured at −0.45 V vs. Ag/AgCl afteraddition of concentrations of 2, 5, 10, 15 and 20 mmol dm⁻³ rutheniumhexaamine in 0.1 mol dm³ Tris buffer at pH 9 containing 0.1 mol dm⁻³KCl. Results are shown in FIG. 12.

Example 2b

Amperometric current was measured 1 second after the application of a−0.50 V vs. Ag/AgCl potential step after addition of concentrations of2, 5, 10, 15 and 20 mmol dm⁻³ ruthenium hexaamine in 0.1 mol dm⁻³ Trisbuffer at pH 9 containing 0.1 mol dm⁻³ KCl. Results are shown in FIG.13.

Example 2c

Cyclic voltammetric current was measured at 0.15 V vs. Ag/AgClimmediately after addition of 2, 4, 6, 8 and 10 mmol dm⁻³ NADH in 0.1mol dm⁻³ Tris buffer at pH 9 containing 0.1 mol dm⁻³ KCl to electrodeson which 0.2 mL of a solution containing 0.2 mol dm⁻³ rutheniumhexaamine and 650 KU/mL putadiaredoxin reductase has been dried. Resultsare shown in FIG. 14.

Example 2d

Amperometric current was measured 1 second after the application 0.15 Vvs. Ag/AgCl on the addition of 2, 4, 6, 8 and 10 mmol dm⁻³ NADH in 0.1mol dm⁻³ Tris buffer at pH 9 containing 0.1 mol dm⁻³ KCl to electrodeson which 0.2 mL of a solution containing 0.2 mol dm⁻³ rutheniumhexaamine and 650 KU/mL putadiaredoxin reductase has been dried. Resultsare shown in FIG. 15.

Example 2e

Amperometric current was measured 60 seconds after the application of a0.20 V vs. Ag/AgCl potential step after addition of concentrations of 2,5, 7.5, 10, 12.5 and 15 mmol dm⁻³ glycerol in 0.1 mol dm⁻³ Tris bufferat pH 9 containing 0.1 mol dm⁻³ KCl obtained at electrodes on which 0.3mL of a solution containing 150 U/mL glycerol dehydrogenase, 100 mmoldm⁻³ of NAD, 100 mmol dm⁻³ of ruthenium hexaamine, 100 mmol dm⁻³ ofammonium sulphate, 100 mmol dm⁻³ of potassium chloride has been dried.Results are shown in FIG. 16.

Example 2f

Ratio of amperometric current was measured 60 seconds after theapplication of a −0.50 V vs. Ag/AgCl potential step after addition ofconcentrations of 2, 5, 7.5, 10, 12.5 and 15 mmol dm⁻³ glycerol in 0.1mol dm⁻³ Tris buffer at pH 9 containing 0.1 mol dm⁻³ KCl obtained atelectrodes on which 0.3 mL of a solution containing 150 U/mL glyceroldehydrogenase, 100 mmol dm⁻³ of NAD, 100 mmol dm⁻³ of rutheniumhexaamine, 100 mmol dm⁻³ of ammonium sulphate, 100 mmol dm⁻³ ofpotassium chloride has been dried. Results are shown in FIG. 17.

Example 3

Electrodes were constructed from a 250 μm PET layer on which a 7 μmCoates carbon ink 26-8203 layer had been screen-printed followed by a 30μm Ronseal layer. This layer was punched to produce a 1 mm diameterhole. A base layer was formed by printing a 10 μm Ag/AgCl layer onto a125 μm PET base layer. The base layer was then adhered to the punchedlayer using ARcare 7841 sheet adhesive. Various tests were carried outusing this electrochemical cell which are described in Examples 3a and3b below.

Example 3a

Amperometric current was measured 120 sec after the application of a−0.25 V vs. Ag/AgCl potential step. Showing the effect of additions of1.5, 2.25, 3.0, 4.5, 6.0 mmol dm⁻³ cholesterol to a solution comprising1 KU/mL cholesterol oxidase, 200 KU/mL horseradish peroxidase, 33 mmoldm⁻³ potassium ferrocyanide in 0.1 mol dm⁻³ potassium phosphate bufferat pH 7.4 containing 0.1 mol dm⁻³ KCl to electrodes with a commoncounter/reference electrode configured at the bottom of the well.Results are shown in FIG. 18.

Example 3b

Amperometric current was measured 120 sec after the application of a−0.25 V vs. Ag/AgCl potential step. Showing the effect of additions of1.5, 2.25, 3.0, 4.5, 6.0 mmol dm⁻³ cholesterol to a solution comprising1 KU/mL cholesterol oxidase, 200 KU/mL horseradish peroxidase, 33 mmoldm⁻³ potassium ferrocyanide in 0.1 mol dm⁻³ potassium phosphate bufferat pH 7.4 containing 0.1 mol dm⁻³ KCl to electrodes with a commoncounter/reference electrode configured on the top of the strip. Resultsare shown in FIG. 19.

Example 4

Electrodes were constructed from a 250 μm PET layer on which a 7 μmErcon carbon ink G449C layer had been screen-printed followed by a 30 μmErcon E65615-116D dielectric layer. This was then punched to produce a 1mm diameter hole. A 125 μm PET base layer was coated with a commonAg/AgCl counter reference layer (using Ercon E6165-128). The base layeras formed was then adhered to the punched layer using heat lamination.

Amperometric current was measured 1 second after the application 0.15 Vvs. Ag/AgCl on the addition of 2, 4, 6, 8 and 10 mmol dm⁻³ NADH in 0.1mol dm⁻³ Tris buffer at pH 9 containing 0.1 mol dm⁻³ KCl to electrodeson which 0.2 mL of a solution containing 0.2 mol dm⁻³ rutheniumhexaamine and 650 KU/mL putadiaredoxin reductase has been dried. Resultsare shown in FIG. 20.

1. An electrochemical cell in the form of a receptacle, said cellcomprising a counter electrode and a working electrode, wherein theminimum distance between the working electrode and the counter electrodeis 50 μm, wherein at least one electrode is a micro-electrode having onedimension of less than 50 μm and one dimension of greater than 50 μm,wherein the working electrode is in a wall of the receptacle, andwherein the receptacle contains an electro-active substance, and whereinthe receptacle is shaped so as to restrict movement of theelectro-active substance away from the electrodes when a sample flowsover the electrochemical cell.
 2. An electrochemical cell according toclaim 1, wherein the working electrode is in the form of a continuousband.
 3. An electrochemical cell according to claim 1, which furthercomprises a reference electrode.
 4. An electrochemical cell according toclaim 1, wherein the ratio of the surface area of the counter electrodeto the surface area of the working electrode is at least 25:1.
 5. Anelectrochemical cell according to claim 1, wherein the working electrodeis a micro-electrode having at least one dimension in the range of 0.1to 20 μm.
 6. An electrochemical cell according to claim 1, wherein thereceptacle has a width, or in the case of a cylindrical receptacle adiameter, of from 0.1 to 5 mm.
 7. An electrochemical cell according toclaim 1, wherein the minimum distance between the counter electrode andthe working electrode is from 50 to 1000 μm.
 8. An electrochemical cellaccording to claim 1, wherein a base and/or a wall or walls of thereceptacle contain one or more air-outlet channels.
 9. Anelectrochemical cell according to claim 1, wherein the electro-activesubstance has been dried.
 10. An electrochemical cell according to claim1, wherein the electro-active substance comprises an enzyme.
 11. Anelectrochemical cell according to claim 1, wherein the base of thereceptacle comprises an electro-active substance.
 12. An electrochemicalcell according to claim 1, wherein the base of the receptacle comprisesa membrane, said membrane comprising an electro-active substance.
 13. Anelectrochemical cell according to claim 1, wherein the open end of thereceptacle is at least partly covered by a permeable membrane.
 14. Anelectrochemical cell according to claim 13 wherein the permeablemembrane comprises an electro-active substance.
 15. An electrochemicalcell according to claim 1, wherein the receptacle comprises one or morecapillary flow channels through which a sample may enter.
 16. Anelectrochemical cell according to claim 1, wherein the counter electrodeforms at least a part of a base of the receptacle.
 17. Anelectrochemical cell according to claim 15, wherein the receptacle iscovered by a layer containing the counter electrode.
 18. Anelectrochemical cell according to claim 1, wherein the counter electrodeis in a wall or walls of the receptacle.
 19. An electrochemical cellaccording to claim 1 which is suitable for screening liquid samples. 20.A multi-analyte device comprising a plurality of electrochemical cellsaccording to claim
 1. 21. A process for producing an electrochemicalcell according to claim 1, which process comprises the steps of: (a)forming a first part comprising an insulating material which isoptionally coated with a counter electrode layer; (b) forming a secondpart comprising a laminate of a working electrode layer between twolayers of an insulating material; (c) creating a hole in the secondpart; and (d) bonding said first part to said second part to form areceptacle, which process further comprises placing an electro-activesubstance into the receptacle and optionally drying the electro-activesubstance, and wherein the receptacle is shaped so as to restrictmovement of the electro-active substance away from the electrodes when asample flows over the electrochemical cell.
 22. A process according toclaim 21 wherein the second part comprises a laminate of a counterelectrode layer between two layers of insulating material.
 23. A processaccording to claim 21 wherein the first part comprises an electro-activesubstance.
 24. A process according to claim 21, which further comprisesplacing a membrane over at least a part of the open end of thereceptacle.
 25. A process according to claim 21, wherein step (c)comprises forming two or more holes in said second part, in order toform a multi-analyte device.
 26. A process according to claim 21 whichcomprises the steps of: (a) forming a first part comprising aninsulating material; (b) forming a second part comprising a laminate ofa working electrode layer between two layers of an insulating material;(c) creating, in the second part, a hole and a capillary channel toallow a sample to enter said hole; (d) bonding said first part to saidsecond part to form a receptacle; (e) placing an electro-activesubstance into the receptacle and optionally drying the electro-activesubstance; and (f) bonding to the open end of said receptacle a layerwhich is optionally coated with a counter electrode material.
 27. Aprocess according to claim 26, wherein step (c) comprises forming, insaid second part, two or more holes and two or more capillary channelsto allow a sample to enter said two or more holes, and wherein step (e)comprises inserting an electro-active substance, which may be identicalor different, into one or more of the receptacles formed in step (d), inorder to form a multi-analyte device.
 28. A process according to claim21, wherein one or more of the electrodes is formed by screen or inkjetprinting onto a substrate.
 29. A method of electrochemically testing oneor more compounds of a sample, the method comprising the steps of: (a)providing an electrochemical cell in the form of a receptacle, said cellcomprising a counter electrode and a working electrode, wherein theminimum distance between the working electrode and the counter electrodeis 50 μm, wherein at least one electrode is a micro-electrode having onedimension of less than 50 μm and one dimension of greater than 50 μm,wherein the working electrode is in a wall of the receptacle, whereinthe receptacle contains an electro-active substance, and wherein thereceptacle is shaped so as to restrict movement of the electro-activesubstance away from the electrodes when a sample flows over theelectrochemical cell; (b) inserting the sample into the electrochemicalcell; (c) applying a voltage or a current between the working andcounter electrodes; and (d) measuring the resulting current, voltage orcharge across the working and counter electrodes.
 30. A multi-analytedevice comprising a plurality of electrochemical cells as defined inclaim
 13. 31. An electrochemical cell according to claim 1, wherein theratio of the surface area of the counter electrode to the surface areaof the working electrode is about 1:1.
 32. An electrochemical cellaccording to claim 1, wherein the ratio of the surface area of thecounter electrode to the surface area of the working electrode is from1:1 to 25:1.
 33. An electrochemical cell according to claim 1, whereinthe working electrode is a micro-electrode having at least one dimensionof less than 25 μm.
 34. An electrochemical cell according to claim 1,wherein the receptacle has a width, or in the case of a cylindricalreceptacle a diameter, of at least about 1 mm.
 35. A process accordingto claim 21, wherein the step of creating a hole in the second partcomprises a laser drilling step.